Fibroblast growth factor (fgf)

Human amniotic fluid (AF) was obtained by ultrasound-guided amniocentesis performed on pregnant women for routine prenatal diagnosis purposes at gestational ages ranging from the 18th to the 22nd weeks. Using a 22G needle and under ultrasonographic control, 10 ml of AF was aspirated under aseptic conditions. The procedure is considered part of the standard diagnostic work used to rule out major chromosomal and genetic defects in some foetal disorders.
The specimens were centrifuged at 700g for 15 minutes, and the pellets removed and resuspended in 2 ml of DMEM supplemented with 20% FBS.
The total pool of cells was then plated in a 25 cm² flasks. Cells were fed daily with DMEM supplemented with 20% FBS, 5 ng/ml fibroblast growth factor (FGF) (Promega, Milan Italy), 1% glutamine solution, 1% antibiotic-antimycotic, in a 95% humidified air, with 5% CO2 at 37°C for 48 hours; after that they were inspected for cell attachment, the medium was replaced, and non-adherent cells were removed.
The neural differentiation was achieved by adding to the cells suspended in the basic medium and maintained in culture for 10 DIV the following mixed solution: 1mM dbcAMP, 0.5 mM IBMX, 20 ng/ml hEGF, 40 ng/ml bFGF, 10 ng/ml NGF and 10 ng/ml BDNF and monitored, for neural phenotype, by light microscopy at 2, 6 and 10 DIV.

On arrival, skin biopsies were rinsed in 70% ethanol followed by 2 × PBS 1% Antibiotic-Antimycotic solution (Gibco), prior to dissociation of dermal-epidermal junctions by overnight digestion in a Dispase solution (Life Technologies) at 4 °C. The next day, dermis was discarded and epidermis digested in a trypsin/EDTA solution for 20 min at 37 °C. Cellular suspension was passed through a 70 μm filter and melanocytes re-suspended in MCDB 153 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2% fetal bovine serum (FBS; Perbio, Cramlington, UK), 5 μg/ml insulin (Sigma-Aldrich), 0.5 μg/ml hydrocortisone (Sigma-Aldrich), 16 nM tetradecanoylphorbol-13-acetate (TPA) (Sigma-Aldrich), 1 ng/ml fibroblast growth factor (FGF; Promega, Madison, WI, USA), 15 μg/ml bovine pituitary extract (Invitrogen, Waltham, MA, USA) and 10 μM forskolin (Sigma-Aldrich). Melanocytes were maintained at 37 °C in 95% O₂/5% CO₂ atmosphere and supplemented with 0.08% G418 geneticin (20 μg/ml, Invitrogen) for ~2 weeks to eliminate rapidly growing cells (i.e., keratinocytes hence selecting for melanocyte propagation). Once selected, melanocytes were grown in Cascade Biologics 254 medium (ThermoFisher Scientific) with Human Melanocyte Growth Supplement (HMGS, Gibco).